Objective: The aim of our work was to study the blood parameters and bone marrow morphological changes in rats exposed to increased
amounts of heavy metal salts and the effect of vitamin E.

Material and Method: Investigation of bone marrow structural features and blood parameters was performed in sexually mature Wistar male
rats (n=84).

Results: Exposure to increased amounts of heavy metal salts led to the inhibition of erythropoiesis and leukopoiesis, as well as a synchronized
increase in the number of megakaryocytes which was clearly reflected in the blood: the number of erythrocytes, leukocytes and Hb decreased,
and the number of platelets increased. These changes in the blood and bone marrow were less pronounced when vitamin E was used as an
adjuster.

Conclusion: When increased amounts of HMS enter the rats` bodies, suppression of erythropoiesis and leukocytopoiesis occurs while
thrombocytopoiesis increases. These changes depend on the period of intake of heavy metal salts. The adjustment of vitamin E reduces the
severity of the cytotoxic effect of heavy metals and improves readaptation in the recovery period.

Nowadays hemato-ecological problems are becoming
considerably urgent and research is being conducted in
this area considering the impact of exogenous factors on
biocoenosis. Some of the most powerful pollutants are
heavy metal salts (HMS), and in some districts of the
Sumy region of Ukraine, their concentrations exceed the
maximum permissible range in the water and soil 1.
Unfortunately, relatively little research has been devoted to
the study of the impact of heavy metal compounds (HMC)
on the hematopoietic system despite the rather clear impact
of these factors on the majority of organs 2. Moreover, the
research available on this theme is devoted only to blood, or
only to the hematopoietic system in most cases 3,4.

Qualitative and quantitative blood indicators reflect the
function of the hematopoietic system and the operation of
other internal organs. The influence of pathogenic factors
related to HMS on the process of hematopoiesis may occur
through their direct impact on the bone marrow (BM) or
indirect damage of other internal organs 5. Their negative
effects have been listed as lipid peroxidation induction, competitive replacement of essential trace elements in
the structure of hydroxyapatite, deactivation of enzyme
systems, DNA damage and so on 6. All these negative
impacts can occur in the process of hematopoiesis, and this
is reflected in the peripheral blood.

Recently more attention has been paid to the investigation of
changes in hematopoietic tissue. This has been achieved by
means of trephine biopsy of the iliac bone and preparation
of histological specimens that allow evaluation of the
parenchymal component of bone marrow and changes in
the stroma, which play a regulatory role in the proliferation
and maturation of hematocytes 7.

Another important objective of modern medicine is the
search of adequate corrective and preventive ways that
increase body resistance to the conditions of constantly
growing urbanization and technological progress that lead
to rapid contamination of the environment.

The purpose of our work was the study of the laboratory
parameters of blood and morphological changes of bone
marrow in rats exposed to increased amounts of HMS to investigate the possible correction of the changes with
vitamin E.

The investigation of bone marrow structural features and
blood parameters was performed in sexually mature Wistar
male rats (4 months old, n=84), considering the impact
of the animals` ages on the process of hematopoiesis. All
investigations were performed in accordance with “General
ethical animal experimentation” (Kyiv, 2001) and the
Helsinki Declaration of the General Assembly of the World
Medical Association (2000). Rats were kept on a standard
diet (except some peculiarities in water consumption) in
the vivarium at 20-25ºC temperature, no more than 50%
humidity, and at day/night light mode.

The distribution of the animals into the groups was as
follows:

The first group included three series of rats (12 rats in each
series). The first series was used as control and the animals
drank ordinary water. In the second series, the rats received
aqueous mixture of heavy metal salts in the concentration
appropriate for the Sumy region and containing 5 mg/l zinc
(ZnSO4x7H2O), 1 mg/l copper (CuSO4x5H2O), 10 mg/l
iron (FeSO4), 0.1 mg/l manganese (MnSO4x5H2O), 0.1
mg/l lead (Pb(NO3)2), and 0.1 mg/l chromium (K2Cr2O7)
1. In the third series, the rats received the same heavy
metal nsalts mixture with vitamin E correction (9.1 mg/kg
of 10% oil oral solution). The animals` dosage calculation
was based on the average daily therapeutic dose for adults.
To examine the effects of subacute and chronic exposure to
HMS on the blood and hematopoiesis, 6 animals in each
series of the first group were removed from the experiment
by decapitation under ether anesthesia on the 30th and
90th days of the study.

The second group, including four series of laboratory
animals with 12 rats in each series, was used to investigate
the readaptation process. The first series was control. The
second included rats that had been consuming a mixture
of heavy metal salts solution for 90 days and then moved
to ordinary drinking water. In the third series, the rats had
been consuming a mixture of heavy metal salts solution with
vitamin E correction for 90 days and had then been moved
to ordinary drinking water and continued to use vitamin
E. In the fourth series, the animals had been consuming a
mixture of heavy metal salts solution for 90 days and had
then started to use ordinary drinking water with vitamin E.
The process of rapid and remote readaptation was examined
by taking 6 animals in each series out of the experiment on
the 30th and 90th days.

Blood obtained from the rats` aorta served as the material
for laboratory research in which the number of erythrocytes,
leukocytes, platelets, hemoglobin (Hb), erythrocyte
sedimentation rate, and the levels of Ca, Na, K, creatinine
and urea were determined when the rats were taken out of
the experiment.

The study of structural bone marrow peculiarities was
performed using the femur. The material was fixed in
10% buffered formalin solution. Decalcification had
been conducted with EDTA solution (pH 7.0) for 14
days, with daily change of the solution. After standard
tissue processing, 4μ-thick sections were taken from
paraffin-embedded tissues and stained for H&E. In order
to differentiate erythropoietic and leukopoietic lines
of hematopoiesis, immunohistochemical studies were
performed to determine the receptors to myeloperoxidase,
CD3 and CD79α. Unfortunately, cytological examination
and quantitative assessment of the stromal component
could not be performed due to the technical difficulties.

The measurement of the size of microspecimen constituent
elements was conducted in the environment of the
«Digimizer» morphometric program (Figure 1). Obtaining
and storage of the images was carried out by means of
the digital image output system «SEO Scan» (Ukraine).
Statistical calculations were performed using Microsoft
Excel 2010 and Attestat 12.0.5. Non-parametric tests
were performed with the Mann-Whitney U test while the
Spearman correlation test was used in correlation analyses.
The results were considered statistically reliable at a
probability level of more than 95% (p <0.05).

Both upwards and downwards variations in blood
test indicators were found (Figure 2), establishing the
dynamic changes in blood under the influence of HMS
and their adjustment with vitamin E, as the duration of
the experiment was prolonged. Blood counts showed
the decrease in erythrocytes, leukocytes and Hb, and the
increase of platelets while rats were receiving HMS.

Figure 2: Indicators of rats’ complete blood
counts. Exp: the period of the experiment,
Read: the period of readaptation.
Blue line: control group, red line: rats
that drank water with HMS, green line:
rats that drank water with HMS and were
administered vitamin E, violet line: rats
that were administered vitamin E during
rehabilitation after HMS.

After day 30, Hb decreased by 11% (p<0.01) and the
number of erythrocytes by 23.3% (p<0.01) in rats of the
second series of the first group. There was a 4.6% increase in
the number of leukocytes and 2.5% increase in the number
of platelets but were not found to be significant (p=0.11 and
p=0.08, respectively). With prolonged duration of receiving
exogenous pollutants (on day 90), a consistent decrease in
Hb levels and in the number of leukocytes and erythrocytes
was observed (18.4%, 32.5%, 15.3%, respectively) while platelets were increased by 11% (p<0.01). The changes were
not so impressive in the blood of rats in the third series of
the first group. On the 90th day, Hb indicators decreased by 6.6%, the number of erythrocytes by 23.3% and leukocytes
by 13.2%, while the number of platelets conversely increased
by 8.2% (p<0.01).

In the readaptation process, blood indicators gradually
normalized, but did not reach the levels of animals in the
control group. The tempo of this process also depended
on the conditions of readaptation. Thus, in rats of the
second series of the second group, increases in Hb levels
and erythrocyte and leukocyte numbers were observed
(12.6%, 35.9%, 8.4%, respectively), while platelet numbers
decreased by 5.3% (p<0.01) on the 90th day. Increases in Hb
levels and erythrocyte and leukocyte numbers were 17.5%,
39.3%, 11.5%, respectively, in animals of the fourth series
of the second group; and 8.9%, 26.3%, 11.3%, respectively,
in rats of the third series of the second group. For these last
two groups, decreases in platelet numbers were found at
rates of 6.8% (p<0.01) and 5.6% (p<0.01), respectively.

The changes in blood biochemical parameters in the rats
of the second and third series of the first group had similar
dynamics (Table I), which was expressed by decreases in
the measured values of Ca (p<0.01) and K (p=0.01) and
by increases of Na (p<0.01), creatinine (p=0.04) and urea
(p<0.01). During readaptation, these indicators gradually
normalized, but in most cases they did not reach the values
of animals in the control group. The speed of recovery
depended on readaptation conditions (whether adjustment
with vitamin E was present or absent) and readaptation
duration.

In the study of morphological features of bone marrow
structure, we found that it undergoes quantitative and
qualitative changes during the experiment, both at the level
of the epiphysis and the diaphysis (Figure 3,4). A gradual decrease in the number of erythropoietic and leukopoietic
cells with simultaneous increase in the number of
megakaryocytes was taking place (control group indicators
were 5.25±1.42%, 14.75±1.42%, 0.16±0.05% at the
epiphysis level and 17.83±0.75%, 51.17±3.27%, 0.16±0.05%
at the diaphysis level, respectively (8)), which reached
maximum values on the 90th day of the experiment. Thus,
the area of erythropoiesis in the second series of rats was
reduced by 30% at the epiphysis level (p=0.03) and by
17.8% at the diaphysis level (p=0.025) in 3 months; the
area of leukopoiesis decreased by 6.1% (p=0.16) and 13.7%
(p<0.01), respectively. The number of megakaryocytes
increased by 27%.

Figure 3: Indicators of bone marrow area that are engaged in various pathways of hematopoiesis in rats.
Exp: The period of the experiment, Read: The period of readaptation, C: indicators in the control series of rats, Blue Line: Rats that
drank water with HMS, Red Line: Rats that drank water with HMS and were administered vitamin E, Green Line: Rats that were
administered vitamin E during rehabilitation after HMS.

Figure 4: Longitudinal section of rat femur on diaphysis. 1) Control group, 2) Rats of the second series of the first group (30th day),
3) Rats of the second series of the first group (90th day). (A: H&E; x400, B: Immunohistochemical determination of myeloperoxidase
receptors; ×400).

Adipose tissue and sinusoids crowded with blood replaced
the area being released from hematopoietic tissue during
the experiment (normally they accounted for 11±2% and
18.7±2.1%, respectively) (8).

Hematopoiesis indicators gradually returned to normal
during readaptation, but they did not reach the values of
the control group of animals (Figure 5). The speed and the
completeness of recovery depended on the use of vitamin E
for adjustment. In rats of the second series, erythropoiesis
increased by 35.6% and 18.1% (p=0.01) respectively in the
femur segment, leukopoiesis increased by 9.5% (p=0.045)
and 8.4 % (p=0.01), and thrombopoiesis decreased by 18%
on the 90th day of readaptation. In animals of the third
series, the area occupied by erythropoiesis increased by
29.5% (p=0.01) at the epiphysis level and by 18.5% (p<0.01)
at the diaphysis level. Leukopoietic areas increased by 5.8% (p=0.03) and 11.4% (p<0.01) respectively, and the number
of platelets decreased by 23%. Indicators of the third series
of animals were as follows: erythropoiesis increased by
35.5% and 18.9% (p=0.01) and leukopoiesis by 10.5%
(p=0.03) and 9.4% (p=0.01), and the number of platelets
decreased by 36%. The area occupied by predecessors of Tand
B-lymphocytes (CD3 and CD79α receptors expression)
did not show any statistically significant changes (p>0.05)
during the experiment and readaptation and it was about
5%.

Figure 5: Longitudinal section of rat femur on the diaphysis. 1) Control group, 4) Rats of the second series of the first group (30th day),
5) Rats of the second series of the first group (90th day). (A: H&E; x400, B: Immunohistochemical determination of myeloperoxidase
receptors; x400).

Comparing the area occupied by leukocyte predecessors
to the area of erythropoiesis (the myeloid/erythroid (M:E)
ratio), a gradual increase was established (due to more rapid
suppression of erythropoiesis) from 1:2.8 (normal) to 1:3.46
(in the 90 days of the experiment). During readaptation, we
have observed some optimization of the M:E ratio (1:2.75),
but it did reach normal values due to the lack of complete
recovery of hematopoiesis.

Laboratory and morphological parameters of the bloodforming
system were monitored during the whole period
and we established a direct correlation between the number
of erythrocytes and erythropoiesis area (r=0.71, p<0.001 for
the epiphysis and r=0.67, p<0.001 for the diaphysis), as well
as between the number of leukocytes and the leukopoietic area (r=0.7, p<0.001 and r=0.87, p<0.001, respectively)
and the platelet levels and the number of megakaryocytes
(r=0.47, p<0.001). Hb values and their sensitivity were
identical to erythrocytes: as the erythropoiesis area became
smaller, the rates of Hb decreased (r=0.47, p<0.001 and
r=0.36, p=0.002 in the appropriate topography of the
femur).

Among the qualitative changes in rats’ bone marrows
during the experiment, we observed hemorrhages, focal
adiposis (due to inhibition of hematopoiesis), myxomatosis
phenomena, sinusoidal ectasia, histiocytic infiltration,
degenerative changes (atrophy, necrosis and apoptosis
phenomena) and others (Figure 6). The intensity of these
changes and the rate of their recovery depended on the
adjustor availability - changes developed slowly and not in
full extent when using vitamin E.

The bone marrow undergoes significant changes during
vital activity, which is associated with age characteristics
and the impact of the environment 4,9. The study was
carried out on 84 rats to determine the impact of increased
amounts of HMS on the hematopoietic system. We found
that exposure to exogenous pollutants led to the inhibition of erythropoiesis and leukopoiesis and synchronized
growth in the number of megakaryocytes which was
clearly reflected in the blood: the number of erythrocytes,
leukocytes and Hb decreased, and the number of platelets
increased. These changes are associated with the direct and
indirect hematotoxic influence of HM, directly affecting
the function of hematopoietic cells (increased lipid
peroxidation and the formation of active oxygen forms, the
DNA destabilization, deactivation of enzyme systems) 6.
HM suppresses the proliferative activity of cells and blocks
their maturation. The estrogen-resembling activity of HM
plays a significant role in the inhibition of hematopoiesis
10, which is reflected in a normal blood test (erythrocytes
and Hb indicators of men are higher and platelet indicators
are lower compared to women). Therefore, the increase in
the number of HM in the blood enhances their estrogenresembling
effect on the bone marrow, considering the presence of estrogen receptors in hematopoietic cells 11.
We do not rule out a direct cytotoxic effect of metals on
blood corpuscles 3. Reactive thrombocytosis is also
associated with slowly progressive anemia, and as a result,
the rate of maturation of platelet predecessors increases and
their destruction decreases 12. The study showed more
pronounced changes in hematopoiesis - the blood system,
namely during the subacute period of the experiment
(30 days) with a gradual slowing on the 90th day, which
is associated with activation of endogenous adaptive
compensatory processes in rats when their living conditions
change.

As seen from the results of the study, significant changes
in biochemical blood tests (increased urea, creatinine,
dismicroelementosis) occur due to the nephrotoxic effects
of HMS. There are also negative effects on bone tissue and the parathyroid gland (initial decrease of Ca because
of probable inhibition of parathormone synthesis and
subsequent increase in the washout of calcium from the
hydroxyapatite bone structure) 13,14. Indirect effects of
HMS on hematopoiesis are realized precisely because of
increasing metabolic products of nitrogenous bases and
dismicroelementosis.

Erythropoietic tissue was found to be the most sensitive
part of bone marrow. Its inhibition reaches 36.6%
(leukopoiesis is maximally inhibited by 14%), which
manifests itself as a rapidly arising anemia. Anemia can
stimulate thrombocytosis, which is also provoked by heavy
metals. It seems that the least affected part of hematopoiesis
is lymphopoiesis. Other studies are needed to see whether
lymphoid tissue is affected in the other parts of the lymphoid
system, i.e. the thymus, spleen, lymph nodes, etc.

As HMS strongly inducts lipid peroxidation, we used
vitamin E as an adjustment agent that has a strong protective
effect on lipids and DNA as a common antioxidant 15. We
observed the protective effects of vitamin E, as the reparative
changes occurred more fully in vitamin E administered rats.
It is also stated that vitamin E does not have any significant
negative effect on the process of hematopoiesis in mature
rats 16. However, the changes that occurred during the
experiment were not completely eliminated in any case
even with the constant administration of vitamin E. This
finding emphasizes the cumulative properties of exogenous
pollutants. Also, we have not observed any significant
difference in the hematopoiesis recovery rate during
different periods of rehabilitation, indicating the gradual
and slow excretion of HMS.

In conclusion, the bone marrow is very vulnerable and also
very sensitive to the variability of living conditions, which
leads to both a parenchymal and stromal reaction. Increased
amounts of heavy metal salts oppress erythropoiesis and
leukocytopoiesis in rats while thrombotcytopoiesis occurs,
as demonstrated in our study. The level of qualitative and
quantitative destructive changes depends on the direct and
indirect effects of exogenous pollutants. More pronounced
changes were observed during the subacute period of the
experiment (first 30 days) with gradual slowing on the 90th
day, which is associated with activation of endogenous
adaptive-compensatory processes in rats when living
conditions change.

The adjustment of vitamin E reduces the severity of the
cytotoxic effect of heavy metals and improves readaptation
in the recovery period. However, even with constant use of
vitamin E, the full range of morphological changes of the
bone marrow does not reach normal levels, which reflects
the cumulative properties of heavy metal salts in the body.